1. Field of the Invention
This invention relates to the field of electrophoresis. More particularly, it concerns an improved process and apparatus for carrying out and detecting electrokinetic separations in open-tubular capillaries.
2. Description of Background Information
In 1974, Pretorius, et al. (J. Chromatogr., 99, 23) described the concept of electroosmosis which they stated to be the flow of a liquid, in contact with a solid surface, under the influence of a tangentially applied electric field. They attributed the electroosmotic flow to the formation of an electric double layer, at the solid-liquid interface, due to the preferential adsorption of ions on the surface. This transport process can be visualized with reference to FIG. 1. In FIG. 1, a small bore double open-ended tube 10 is shown in cut away cross section. The tube is filled with a conductive liquid 11 sometimes referred to herein as a "support electrolyte". The wall of tube 10 contains preferentially adsorbed positive ions 12. (Depending upon the material of tube 10, the adsorbed charge could be negative, instead.) Positive ions 12 attract anions 13 from conductive liquid 11 and set up an electric double layer 14. This preferential attraction of anions to the wall results in a net excess positive charge in the body of liquid 11. Thus, when an electric potential is applied, such as a 30 kV potential between electodes 15 and 16, located at the ends of the column of liquid 11 contained within tube 10, the positively charged liquid moves toward the cathode. Pretorius et al. proposed the use of this process in thin-layer and high speed liquid chromatography settings.
In 1979, Mikkers, et al. (J. Chromatogr. 169, 11) described the use of narrow-bore (e.g. 0.2-0.35 mm i.d.) tubes for high performance zone electrophoresis. More recently, J. W. Jorgenson and K. D. Lukacs have reported (J. Chromatog. 218 (1981), 209; Anal. Chem. 53 (1981), 1298; and Science 222 (1983), 266) the use of 75 .mu.m glass capillaries to carry out such separations. Tsuda, et al, reported similar work in J. Chromatog. 248 (1982), 241 and J. Chromatog. 264 (1983), 385. An advantage to the use of capillary channels is that joule heating effects which disturb the sample flow are minimized.
The separation process relies upon the electroosmosis effect just described and upon the differential effect of the electric field on solutes in the liquid medium depending upon their positive, neutral or negative charge. These related effects may be visualized with reference to FIG. 2. FIG. 2 is a copy of FIG. 1 but with various charged species 18 and 19 in liquid 11. Cationic species 18 is electrophoretically drawn toward cathode 16. Anionic species 19 is electrophoretically repelled by cathode 16. As is shown in FIG. 2, and as is usualy the case, the velocity of the liquid 11 is larger than the electrophoretic velocities of the species in solution such that all the species can be seen to move in the direction of the electroosmotic flow but at differing rates. The combination of electroosmotic flow and electrophoretic movement is referred to in the literature and herein as electrokinetic movement, and a separation which relies upon these two effects is referred to as an electrokinetic separation.
Moreover, electroosmotic flow has plug flow characteristics as opposed to laminar flow characteristics. This favors high resolution separations. One can, in theory, use an electrokinetic separation to provide separation of species in solution, and one should in principle be able to detect these separations. However, as stated by Jorgenson and Lukacs in the conclusion of their Science review article, "The greatest obstacle to further development and utilization of capillaries [in such separation methods] is the requirement of extremely sensitive detection, and more types of detection with higher sensitivity are greatly needed." The types of detectors used heretofore to indicate the presence of species as they move through electrokinetic separation columns have included UV absorption and conductivity used by Mikkers, et al; on-column fluorescence detection with lamp excitation used by Jorgenson and Lukacs and UV absorption detection used by Tsuda, et al. David, et al, of the Oak Ridge National Laboratory in research report ORNL/TM-9141 in contract W-7405-eng-26 have disclosed an on-column lamp-excited fluorescence detector system and its use in connection with a capillary electrophoresis system. The selection of a suitable detection system is rendered more difficult by the practical consideration of operator safety when high voltages are present. With electric potentials in the range of several tens of thousands of volts passing through the sample as it is being measured, the detector must be reliable and require no operator manipulation in or directly around the sample.
It is an object of the present invention to provide an improved detection method and system sought by the art. It is a further object of this invention to provide new and more sensitive electrokinetic assay methods by employing this improved detection method and system.